CN111781181B - Detection method of porous silicon Bragg reflector biosensor - Google Patents
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract
The invention discloses a detection method of a porous silicon Bragg reflector biosensor. The first signal light is probe light with 635nm wavelength reflected from the surface of the porous silicon bragg reflector. The biological probe is marked by semiconductor quantum dots and then reacts with target molecules in the porous silicon Bragg reflector, and the semiconductor quantum dots play a role in refractive index amplification. The specific binding of biomolecules within the porous silicon bragg mirror causes an increase in the refractive index of the device resulting in an increase in the detection of reflected light. The second signal light is the fluorescence of the quantum dots in the biological reactant. The quantum dots in the reactant are excited by short wavelength light to generate fluorescence with the wavelength of about 630 nm. The fluorescence signal is further enhanced by a porous silicon bragg mirror. The digital microscope is used for simultaneously obtaining two light superimposed images on the surface of the porous silicon Bragg reflector, and the target biomolecules can be detected with high sensitivity by calculating the average gray value change of the images before and after biological reaction.
Description
Technical Field
The invention relates to the technical field of biosensors, in particular to a detection method of a porous silicon Bragg reflector biosensor.
Background
The porous silicon is a nano-structure material with the advantages of large specific surface area, good bioactivity, good biocompatibility, luminescence at room temperature and the like, can be prepared into devices with various optical structures, can enhance and amplify optical signals, and is applied to various optical biosensors, such as microcavity sensors, bragg reflector sensors, grating coupling waveguide sensors and the like.
The detection mechanism of the porous silicon optical biosensor mainly comprises two types, wherein the first type is to detect the refractive index change caused by biological reaction, obtain the refractive index change of the porous silicon biosensor before and after the biological reaction, and further determine the concentration of biomolecules according to the refractive index change. The second type is to detect the fluorescence change caused by biological reaction, detect the change of fluorescent label of biological molecule, and then determine the concentration of biological molecule by the change of fluorescent label. In order to further improve the detection sensitivity of the porous silicon optical biosensor, semiconductor colloid quantum dots are introduced. The semiconductor colloid quantum dot has the advantages of good light stability, long fluorescence life, controllable surface characteristics and the like, and has good biocompatibility after surface modification. In the first type of method, the change in refractive index caused by a biological reaction is amplified by quantum dot labeling of a biomolecular probe. In the second class of methods, the biomolecular probe is labeled with quantum dots as fluorescent labels.
The digital image method is used for detecting biomolecules or biochips by the porous silicon biosensor, solves the problem that the detection sensitivity is limited when the biological detection is carried out by utilizing an angle spectrometry in the detection based on refractive index change, and can eliminate measurement errors caused by instability of detection laser and a light detector by taking a unit image which does not generate biological reaction as a calibration reference.
Accordingly, a detection method combining a digital image method with two porous silicon optical biosensor detection methods based on refractive index change and fluorescence change is provided, which enables low-cost and high-sensitivity biological detection.
Disclosure of Invention
The invention aims to provide a detection method of a porous silicon Bragg reflector biosensor, which aims to solve the problems in the background technology.
In order to achieve the above purpose, the present invention provides the following technical solutions: the detection method of the porous silicon Bragg reflector biosensor comprises the following specific steps:
S1: the method comprises the steps of using P-type monocrystalline silicon, cutting a silicon wafer into small pieces, preparing a porous silicon area to be prepared into a round shape, and preparing a porous silicon Bragg reflector by a single-groove anode electrochemical corrosion method;
S2: the prepared porous silicon Bragg reflector is soaked in hydrogen peroxide solution for oxidization, so that a SiO 2 layer is formed on the surface of the porous silicon Bragg reflector; taking out, repeatedly washing with deionized water and absolute ethyl alcohol, and cooling at room temperature; then soaking the oxidized sample in 3-aminopropyl triethoxysilane to form amino; taking out, repeatedly washing with deionized water and ethanol, blow-drying at room temperature, and placing into a vacuum drying oven; immersing the silanized porous silicon sample into glutaraldehyde water solution for soaking after taking out; taking out, and repeatedly flushing with phosphate buffer and deionized water to remove excessive glutaraldehyde;
S3: diluting target DNA with PBS to six concentrations, and modifying the porous silicon sample with 6 different concentrations of target DNA; placing the sample in an incubator, taking out the sample, flushing with PBS, flushing out redundant DNA which is not connected to the porous silicon sample, and drying the DNA in N 2; soaking in ethanolamine hydrochloride solution; placing the mixture into an incubator, taking out the mixture, and thoroughly flushing the mixture by using HEPES buffer;
S4: taking the quantum dots by using a micropipette, dripping the quantum dots into a micropipette, adding PBS, stirring, adding EDC and sulfo-NHS to activate the quantum dots, carrying out concussion reaction, adding amino-modified probe DNA, and carrying out concussion reaction in a dark place; taking out, centrifuging in a centrifuge, and removing supernatant and remaining sediment;
S5: dripping the solution of the quantum dot coupled probe DNA to the surface of porous silicon connected with target DNA molecules with different concentrations by using a micropipette, reacting at constant temperature to hybridize the DNA, taking out, repeatedly flushing with PBS and DI to remove excessive DNA and QDs, and drying in N 2;
S6: connecting the porous silicon sample into a detection light path of at most Kong Guibu Bragg biosensors, and exciting fluorescence of the porous silicon sample by reflected light and excitation light of the detection light, and simultaneously entering a digital microscope through a filter;
S7: obtaining digital images before and after biological reaction of 6 kinds of DNA with different concentrations and probe DNA coupled with quantum dots by using a digital microscope, and calculating average gray values of the digital images before and after biological reaction of the 6 kinds of DNA with different concentrations and probe DNA coupled with quantum dots;
s8: the concentration of the biomolecule to be measured is calculated by calculating the average gray value variation before and after the biological reaction.
Preferably, in S1, the resistivity of the P-type monocrystalline silicon is 0.01-0.06 Ω & cm, the crystal orientation is <100>, and the thickness is 400+ -10 μm; cutting the silicon wafer into 1.5X1.5 cm pieces; the diameter of the circle of the porous silicon area is 0.8cm; in the single-groove anode electrochemical corrosion method, the electrolytic corrosion solution comprises the following components in volume ratio of 1:1, the concentration of hydrofluoric acid is 40 percent and the concentration of absolute ethanol is more than or equal to 99 percent; the current density of the electrochemical corrosion of the porous silicon Bragg reflector is 40mA/cm 2、110mA/cm2, and the corresponding corrosion time is 1.1s and 0.9s.
Preferably, the porous silicon Bragg reflector in the step S2 is put in a 30% hydrogen peroxide solution at 60 ℃ for 3 hours of oxidation; the concentration of 3-aminopropyl triethoxysilane is 5%, and the composition of the 3-aminopropyl triethoxysilane is that of amino propyl triethoxysilane: methanol: deionized water = 1:10:10, soaking time is 1h; the internal temperature in the vacuum drying oven is 100 ℃, and the drying time is 10min; the concentration of the silanized porous silicon sample immersed in the glutaraldehyde aqueous solution was 2.5%, and the immersion time was 1h.
Preferably, in S3, the internal temperature of the two thermostats is 37 ℃, the operating time of the former thermostats is 2 hours, and the operating time of the latter thermostats is1 hour.
Preferably, the amount of quantum dots in S4 is 50 μl,8 μΜ; the microcentrifuge tube is 1.5ml in size; the amount of PBS added was 340uL; stirring for 5min; EDC was added in an amount of 30. Mu.L, 0.01M; the addition amount of sulfo-NHS is 30 mu L and 0.01M; the shaking reaction time is 10min; the concentration of the amino-modified probe DNA was 10. Mu.M, and the addition amount was 200. Mu.L; the light-shielding concussion reaction time is 10 hours; the parameters of the centrifugal machine are 10000r/min, and the centrifugal time is 10min.
Preferably, the amount of the solution of the quantum dot coupled probe DNA in S5 is 50. Mu.L; the temperature of the constant temperature reaction was 37℃for 2 hours.
Compared with the prior art, the invention has the beneficial effects that: a method for detecting a porous silicon Bragg reflector biosensor includes preparing a porous silicon Bragg reflector by an electrochemical corrosion method, sequentially carrying out thermal oxidation treatment, silanization treatment and glutaraldehyde treatment, marking a biological probe by carboxyl water-soluble CdSe/ZnS quantum dots, then reacting with target molecules in the porous silicon Bragg reflector, using a red light semiconductor laser with a wavelength of 635nm as a detection light source, using an ultraviolet semiconductor laser with a wavelength of 375nm as an excitation light source, exciting quantum dots in the reactant in porous silicon holes of a Bragg structure to generate fluorescence, enabling fluorescent signals to be enhanced through the Bragg reflector, enabling reflected light to be enhanced after biological reaction of the biological molecules in the porous silicon, obtaining digital images before and after biological reaction of two light superimposed images on the surface of the porous silicon Bragg reflector by a digital microscope, and detecting the concentration of the target biological molecules by calculating the average gray value change of the digital images before and after biological reaction.
Drawings
FIG. 1 is a graph showing the reflection spectrum of a theoretical simulation Bragg reflector with the effective refractive indexes increased by 0.005, 0.01 and 0.02;
FIG. 2 is a graph of the theoretical simulated reflectance variation with effective refractive index of a Bragg reflector according to the present invention;
FIG. 3 is a reflection spectrum of a theoretical simulation porous silicon Bragg reflector with incidence angles of 0 DEG, 5 DEG and 10 DEG;
FIG. 4 is a detection light path of the porous silicon Bragg biosensor of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to fig. 1-4, the present invention provides a technical solution: the detection method of the porous silicon Bragg reflector biosensor comprises the following specific steps:
S1: using P-type single crystal silicon (resistivity 0.01-0.06 Ω & cm, crystal orientation <100>, thickness 400+ -10 μm), the wafer was cut into 1.5X1.5 cm pieces, and the porous silicon region to be produced was circular with a diameter of 0.8cm. The porous silicon Bragg reflector is prepared by a single-groove anode electrochemical corrosion method, and the electrolytic corrosion solution comprises the following components in percentage by volume: 1 (concentration: 40%) and absolute ethanol (C 2H5 OH, concentration: 99%). The current density of the electrochemical corrosion of the porous silicon Bragg reflector is 40mA/cm 2、110mA/cm2, and the corresponding corrosion time is 1.1s and 0.9s.
S2: the prepared porous silicon Bragg reflector is soaked in 30% hydrogen peroxide solution, is put in a 60 ℃ environment for oxidation for 3 hours, so that a SiO 2 layer is formed on the surface of the porous silicon Bragg reflector, is taken out, is repeatedly washed by deionized water and absolute ethyl alcohol, and is placed at room temperature for cooling. Then placing the oxidized sample into 5% of 3-aminopropyl triethoxysilane (aminopropyl triethoxysilane: methanol: deionized water=1:10:10), soaking for 1h to form amino groups on the sample, taking out, repeatedly washing with deionized water and ethanol, blow-drying at room temperature, and placing into a vacuum drying oven at 100 ℃ for 10min. The silanized porous silicon sample is immersed in 2.5% glutaraldehyde solution for 1h, taken out and repeatedly washed with Phosphate Buffer Solution (PBS) and deionized water to remove the excess glutaraldehyde.
S3: the target DNA was diluted to six concentrations with PBS, the porous silicon samples were modified with 6 different concentrations of target DNA, placed in a 37℃incubator for 2h, removed, rinsed with PBS, rinsed to remove excess DNA not attached to the porous silicon samples, and dried in N 2. Soaking in 3M ethanolamine hydrochloride solution, placing in a 37 deg.C incubator for 1 hr, taking out, and thoroughly washing with HEPES (PH 9) buffer solution.
S4: 50 mu L of 8 mu M quantum dots are dripped into a 1.5mL microcentrifuge tube by using a micropipette, 340 mu L of PBS is added, after stirring for 5min, 30 mu L of 0.01M EDC and 30 mu L of 0.01Msulfo-NHS are added to activate the quantum dots, and the final concentration of the quantum dots is 1 mu M after shaking reaction for 10 min. 200. Mu.L of amino-modified probe DNA was added thereto at a concentration of 10. Mu.M, and the reaction was carried out with shaking in the absence of light for 10 hours. Taking out, centrifuging in 10000r/min centrifuge for 10min, and removing supernatant and precipitate.
S5: 50 mu L of the solution of the quantum dot coupled probe DNA is dripped on the surface of porous silicon which is connected with target DNA molecules with different concentrations by a micropipette, the reaction is carried out for 2 hours at the constant temperature of 37 ℃ to lead the DNA to hybridize, then the DNA is taken out, and the excess DNA and QDs are removed by repeated flushing with PBS and DI, and the DNA is dried in N 2.
S6: in the detection light path of the porous silicon sample connected to the porous silicon Bragg biosensor in FIG. 4, the laser 1 is a 375nm semiconductor laser, M 1 is a plane mirror, lenses L 1 and L 2 form a collimation beam expanding system, BS 1 is a half-reflecting half-lens, the laser 2 is a 635nm semiconductor laser, M 2 is a plane mirror, lenses L 3 and L 4 form a collimation beam expanding system, BS 2 is a half-reflecting half-lens, G is a goniometer, and L 5 is a 600nm long-pass filter. The reflected light and the excitation light of the probe light excite fluorescence of the porous silicon sample while entering the digital microscope through the filter.
S7: obtaining digital images before and after biological reaction of 6 kinds of DNA with different concentrations and probe DNA coupled with quantum dots by using a digital microscope, and calculating average gray values of the digital images before and after biological reaction of the 6 kinds of DNA with different concentrations and probe DNA coupled with quantum dots;
s8: the concentration of the biomolecule to be measured is calculated by calculating the average gray value variation before and after the biological reaction.
The first signal light is probe light with 635nm wavelength reflected from the surface of the porous silicon bragg reflector. The biological probe is marked by semiconductor quantum dots and then reacts with target molecules in the porous silicon Bragg reflector, and the semiconductor quantum dots play a role in refractive index amplification. The enhancement of the detection reflected light is caused by the increase of the refractive index of the device due to the specific binding of biomolecules within the porous silicon bragg mirror. The second signal light is the fluorescence of the quantum dots in the biological reactant. The quantum dots in the reactant are excited by short wavelength light to generate fluorescence with the wavelength of about 630 nm. The fluorescence signal is further enhanced by a porous silicon bragg mirror. The digital microscope is used for simultaneously obtaining two light superimposed images on the surface of the porous silicon Bragg reflector, and the target biomolecules can be detected with high sensitivity by calculating the average gray value change of the images before and after biological reaction.
The high refractive index layer of the porous silicon bragg mirror was assumed to have a refractive index of 1.42, a thickness of 98nm, and the low refractive index layer had a refractive index of 1.12 and a thickness of 124nm. The center wavelength of the porous silicon Bragg reflector was calculated to be 556nm by the transfer matrix method, as shown by the black line in FIG. 1.
The semiconductor laser is perpendicularly incident on the surface of the porous silicon Bragg reflector, so that the light intensity of reflected light is minimized. After the addition of biomolecules, i.e. after increasing the refractive index of each layer of the porous silicon bragg mirror, the spectrum will shift to the right as a whole, and the refractive index increases by 0.005, 0.01 and 0.02 as shown in fig. 1. Fig. 2 shows the relationship between the surface reflectivity of the porous silicon bragg reflector and the effective refractive index of the device, which is calculated by theory.
Furthermore, if the prepared porous silicon Bragg reflector is subjected to thermal oxidation treatment, silanization treatment, glutaraldehyde treatment, target biological molecules and biological probes marked by semiconductor quantum dots, the wavelength corresponding to the weakest light intensity at the photon forbidden band edge of the porous silicon Bragg reflector is larger than 635nm due to the increase of the effective refractive index of the device, and a method for changing the incident angle can be adopted to enable the reflection spectrum to be blue-shifted, so that the position with the minimum reflectivity at the photon forbidden band edge of the porous silicon Bragg reflector is exactly located at 635 nm. Fig. 3 shows reflection spectra for incidence angles of 0 °,5 °,10 °.
Further, the invention adopts a semiconductor laser with 635nm wavelength to generate detection light and a semiconductor laser with 375nm wavelength to generate excitation light, which is used as quantum dots in excitation reactants. The semiconductor laser is used because the semiconductor laser has the advantages of small volume, light weight, long service life, portability and the like, and can be better used for developing practical biological detection products.
Further, the enhancement of the detection reflected light is caused by the fact that the biological reaction between the biological probe marked by the semiconductor quantum dot and the target molecule in the porous silicon causes the effective refractive index of the porous silicon Bragg reflector to be increased, the reflection spectrum of the porous silicon Bragg reflector is red shifted, the reflection spectrum of the porous silicon Bragg reflector is the lowest at 635nm of the original forbidden band edge, the reflection spectrum is red shifted, the reflection spectrum at 635nm is increased, and the reflection light is enhanced.
Furthermore, the fluorescence signal is further enhanced by the porous silicon Bragg reflector, because the porous silicon Bragg reflector does not emit fluorescence before biological reaction, after biological reaction, quantum dots in the reactant are excited in the porous silicon holes of the Bragg structure to generate fluorescence, the porous silicon Bragg reflector can reflect the fluorescence falling in the high reflection band of the porous silicon Bragg reflector, so that the fluorescence emitted downwards is reflected into the fluorescence emitted upwards, and the fluorescence is enhanced by the porous silicon Bragg reflector.
Further, a digital microscope is used for simultaneously obtaining two light superimposed images of the surface of the porous silicon Bragg reflector, and the target biological molecules are detected by calculating the average gray value change of the images before and after biological reaction.
The detection method of the porous silicon Bragg biosensor provided by the invention is quick, efficient, high in detection sensitivity and low in detection cost, and only needs a digital microscope and a common optical device.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (1)
1. A detection method of a porous silicon Bragg reflector biosensor is characterized by comprising the following steps of: the method comprises the following specific steps:
S1: cutting a silicon wafer of the P-type monocrystalline silicon into small pieces by using the P-type monocrystalline silicon, wherein the porous silicon area to be prepared is round, and preparing the porous silicon Bragg reflector by a single-groove anode electrochemical corrosion method;
S2: immersing the prepared porous silicon Bragg reflector in hydrogen peroxide solution for oxidization, so that a SiO 2 layer is formed on the surface of the porous silicon Bragg reflector; taking out, repeatedly washing with deionized water and absolute ethyl alcohol, and cooling at room temperature to obtain a porous silicon sample; then soaking the porous silicon sample in 3-aminopropyl triethoxysilane to form amino groups on the porous silicon sample; taking out, repeatedly washing with deionized water and absolute ethyl alcohol, drying at room temperature, and placing into a vacuum drying oven; immersing the silanized porous silicon sample into glutaraldehyde water solution for soaking after taking out; taking out, and repeatedly flushing with phosphate buffer and deionized water to remove excessive glutaraldehyde;
s3: diluting target DNA by PBS to six concentrations, and modifying the porous silicon sample with 6 different concentrations of target DNA; placing the sample in an incubator, taking out the sample, flushing with PBS, flushing out redundant DNA which is not connected to the porous silicon sample, and drying the DNA in N 2; soaking in ethanolamine hydrochloride solution; placing the mixture into an incubator, taking out the mixture, and thoroughly flushing the mixture by using HEPES buffer; obtaining digital images of 6 target DNA with different concentrations by using a digital microscope, wherein the digital images are used as digital images before biological reaction occurs;
S4: taking the quantum dots by using a micropipette, dripping the quantum dots into a micropipette, adding PBS, stirring, adding EDC and sulfo-NHS to activate the quantum dots, carrying out concussion reaction, adding amino-modified probe DNA, and carrying out concussion reaction in a dark place; taking out, centrifuging in a centrifuge, and removing supernatant and remaining sediment;
S5: dripping a solution of quantum dot coupled probe DNA to the surface of porous silicon connected with target DNA molecules with different concentrations by using a micropipette, reacting at constant temperature to hybridize the DNA, wherein the hybridization of the DNA is that the hybridization reaction is carried out on the target DNA and the probe DNA coupled with the quantum dot, then taking out the probe DNA, repeatedly flushing the probe DNA with PBS and deionized water to remove excessive DNA and QDs which are not hybridized successfully, and drying the probe DNA in N 2; a digital microscope is used for obtaining a digital image after hybridization reaction of the target DNA and the probe DNA coupled with the quantum dots, and the digital image is used as a digital image after biological reaction;
S6: connecting the porous silicon sample to a detection light path of a Kong Guibu Bragg reflector biosensor, and exciting fluorescence of the porous silicon sample by reflected light and excitation light of detection light, and entering a digital microscope through a filter; wherein the detection light is signal light with 635nm wavelength reflected from the surface of the porous silicon Bragg reflector and is used as first signal light; the fluorescence is generated by quantum dots in reactants in porous silicon holes under the excitation of short-wavelength light, and the signal light with the wavelength of about 630nm is used as second signal light;
S7: obtaining digital images before and after biological reaction of 6 kinds of DNA with different concentrations and probe DNA coupled with quantum dots by using a digital microscope, and calculating average gray values of the digital images before and after biological reaction of the 6 kinds of DNA with different concentrations and probe DNA coupled with quantum dots;
S8: simultaneously obtaining an image of the surface of the porous silicon Bragg reflector, which is overlapped by the first signal light and the second signal light, by using the digital microscope, and calculating the concentration of the biomolecule to be detected by calculating the average gray value variation of the images before and after biological reaction, wherein the biomolecule to be detected is the concentration of the unknown DNA molecule to be detected;
Wherein: in S1, the resistivity of the P type monocrystalline silicon is 0.01-0.06 omega cm, the crystal orientation is <100>, and the thickness is 400+/-10 mu m; cutting the silicon wafer into 1.5X1.5 cm pieces; the diameter of the circle of the porous silicon area is 0.8cm; in the single-groove anode electrochemical corrosion method, the electrolytic corrosion solution comprises the following components in volume ratio of 1:1 and absolute ethyl alcohol concentration, wherein the hydrofluoric acid concentration is 40%, and the absolute ethyl alcohol concentration is more than or equal to 99%; the current density is 40mA/cm 2、110mA/cm2 when the porous silicon Bragg reflector is electrochemically corroded, and the corresponding corrosion time is 1.1s and 0.9s;
wherein: oxidizing the porous silicon Bragg reflector in the S2 for 3 hours in a 60 ℃ environment of 30% hydrogen peroxide solution; silanization treatment is carried out on the oxidized porous silicon sample, and the porous silicon sample is placed in a 3-aminopropyl triethoxysilane solution with the concentration of 5% for 1h; the internal temperature in the vacuum drying oven is 100 ℃, and the drying time is 10min; the concentration of the porous silicon sample immersed into the glutaraldehyde aqueous solution after silanization is 2.5%, and the immersion time is 1h;
Wherein: in the step S3, the internal temperature of the two thermostats is 37 ℃, the working time of the former thermostats is 2 hours, and the working time of the latter thermostats is 1 hour;
Wherein: the amount of quantum dots in S4 was 50 μl,8 μΜ; the microcentrifuge tube is 1.5ml in size; the amount of PBS added was 340uL; stirring for 5min; EDC was added in an amount of 30. Mu.L, 0.01M; the addition amount of sulfo-NHS is 30 mu L and 0.01M; the shaking reaction time is 10min; the concentration of the amino-modified probe DNA was 10. Mu.M, and the addition amount was 200. Mu.L; the light-shielding concussion reaction time is 10 hours; parameters of the centrifugal machine are 10000r/min, and the centrifugal time is 10min;
Wherein: the amount of the solution of the quantum dot coupled probe DNA in S5 was 50 μl; the temperature of the constant temperature reaction was 37℃for 2 hours.
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